The present invention relates to a method, system and kit for use in monitoring of DNA expressions within cells.
The following is a list of references which are intended for a better understanding of the background of the present invention:
Groskreutz, D. and Schenborn, E. T., Reporter systems. In: Methods in molecular biology, Vol. 63: Recombinant protein protocols; detection and isolation. R. S. Tuan (ed.), pp. 173-218, Humana Press Inc., Totowa, N.J.
Jain, V. K. and Magrath, I. T., A chemiluminescent assay for quantitation of xcex2-galactosidase in the fentogram range: application to quantitation of xcex2-galactosidase in lacZ-transfected cells, Anal. Biochem., 199:119-124 (1991).
Kulys, J., Razumas, V and Malinauskas, A., Electrochemical oxidation of catechol and p-aminophenol esters in the prsence of hydrolase, J. Electroanal. Chem. [Bioelectrochem. Bioenerg. 7] 116.11-24 (1980).
Masson, M., Liu, Z., Haruyama, T., Kobatake, E., Ikariyama, Y and Aizawa, M., Immunosensing with amperometric detection, using galactosidase as label and p-aminophenyl-xcex2-D-galactopyranoside as substrate. Anal. Chim. Acta 304;353-359 (1995).
Miler, J. H., A short course in bacterial genetics, p. 72-74, Cold Spring Harbor Press., Cold Spring Harbor, N.Y. (1992).
Rosen, I. and Rishpon, J., Alkaline phosphatase as a label for heterogeneous immunoelectrochemical sensor, J. Electroanal. Chem., 258:27-39 (1989).
Silhavy, T. J. and Beckwith, J. R., Uses of lac fusions for the study of biological problems, Microbiol. Rev. 49;398-418 (1985).
U.S. Pat. No. 5,149,629.
Weichart, D., Lang, R., Henneberg, N., and Hengge-Aronis, R., Identification and characterization of stationary phase-inducible genes in Escherichia coli. Mol. Microbiol, 10:407-420 (1993).
Yin, H. H. and Villarejo, M., OsmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli., J. Bacteriol. 174:3637-3644 (1992).
Reporter gene systems are used in gene expression studies and for biosensor developmont, Various analytical methods are available for monitoring the protein expressed by a reporter gene. These methods include photometry, radiometry, fluorescence, colorimetry and immunoassays (Groskreutz, D. et al., 1997). A light emitting gene was also used as the reporter gene and several techniques have been developed to monitor the light emitting from the cells (Legocki, R. P. et al., 1993).
The predominantly used methods for the identification and quantification of the reporter gene products in cell cultures involve repeated samplings of the culture and an assay for the enzymatic activity, that often involves an additional step of lysis or permeabilization of the cell and must be performed under aerobic conditions. These procedures perturb the culture and are time consuming at times, providing results only after several hours.
The gene lacZ, coding for the Escherichia coli enzyme xcex2-galactosidase is one of the most widely used reporter genes (Silhavy and Beckwith, 1985) and has been used in colorimetric assays (Miler, 1992), or using fluorometry or chemilluminometry (Jain and Magrath, 1991). These methods involve the permeabilization of the cells followed by a multi-step procedure.
The enzymatic activity of xcex2-galactosidase can be determined electrochemically by using the substrate p-aminophenyl-xcex2-D-galactopyranoside (PAPG). The product of the enzymatic reaction, p-aminophenol (PAP) is oxidized at an electrode.
Several electroanalytical methods for PAP detection have been reported (Kulys and Malinauskas, 1980; Masson et al, 1995). An electrochemical imunoassay, in which a constant potential is applied on the electrode and the current generated by the oxidation of PAP is measured was also described (U.S. Pat. No. 5,149,629).
In accordance with the present invention novel means are provided for monitoring of gene expression within cells. As will be detailed further below, the invention may be used for determination of cell parameters in a cell culture, for cell biology research, for determining parameters or conditions of a sample, etc. as well as for biosensor development.
In accordance with one aspect, the present invention provides a method for detecting a parameter of a host cell, comprising:
(a) transfecting said cell with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation wit said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) placing the transfected cells in an electrochemical cell; and
(c) measuring the level of the electrical signal, a signal above a threshold level indicating the presence of said parameter.
The present invention also provides a method for determining a parameter of a medium, comprising:
(a) providing cells transfected with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) placing said cells in the medium or a sample thereof; and
(c) providing an electrochemical cell and measuring level of said electrical signal, the level of said signal being correlated to the level of said parameter.
Still further provided by the invention is a method for monitoring a growth status parameter, said parameter being a parameter characteristic of a defined growth or cell cycle phase or a defined culture phase of a cell culture, comprising:
(a) providing a culture of cells, wherein at least some of the cells are transfected with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with the growth status parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) in an electrochemical cell, continuously or periodically measuring said electrical signal, said signal being indicative of said growth status parameter.
By a further of its aspects the present invention provides a system for monitoring expression of a target promoter, comprising:
(a) an electrochemical cell;
(b) cells transfected with an expressible DNA sequence under expression control of said target promoter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement; and
(c) apparatus for measurement of said electrical signal.
By a still further aspect the present invention further provides a kit for use in detecting a parameter of host cell, a medium parameter or a cell or cell culture growth stats indicator parameter, comprising:
(a) host cells transfected wit an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) at least one component of an electrochemical cell, said electrochemical cell being adapted for receiving and holding the host cells and for performing said electrochemical measurement.
The following is an explanation of some terms used above and in the following description and claims:
Parameterxe2x80x94when applied to a cell, referring to a certain property of interest within the cell which may include a certain phase of the cell cycle, a response of the cell to stimulants existing in an extracellular medium or a change in the expression of any gene (such stimulants may include a certain environmental pollutant, toxic chemical nutritional substance, existence of a substance which regulates cell activity or growth, production of certain substances within the cell, etc.). When applied to a mediumxe2x80x94referring to the existence in medium of substances or a condition (e.g. temperature, ionic strength, etc.) which affects cellular parameters in a host cell.
Host cellxe2x80x94a cell which is transfected with a DNA construct in accordance with the invention. Various kinds of host cells may be used as described below.
Enzymatically active productxe2x80x94a product of gene expression which can perform an enzymatic activity. Such a product may be an enzyme per se, typically active within the cell, or may be an enzyme having a signal peptide or protein, (e.g. an expression product having an expressible DNA sequence obtained by fusing a sequence encoding the enzyme and a sequence encoding the signal, peptide or protein).
Electrochemical measurementxe2x80x94a measurement performed by the use of electrodes in a solution, typically in an electrochemical cell. The measurement may be performed, for example, by chrono-amperometry, chrono-potentiometry, cyclicvoltometry, chrono-coulometry or square wave voltometry. A signal detectable in such a measurement, is one that differs in such electrochemical measurement from the control.
Mediumxe2x80x94any medium, which may be a liquid, a solid, or a gas in which a certain quality is to be measured. Such a quality may be the existence of a certain substance in the medium, a temperature of the medium, etc. Where the medium is an aqueous liquid, it can be applied as such onto the host cells. Where the medium is a gas or a solid, it has to be first mixed with a liquid medium which will then be applied onto the host cells. Alternatively, a gas medium may be bubbled through a liquid medium comprising a culture of the host cells. Similarly, a solid substrate, such as soil, may also be admixed with a medium containing a culture of the tested cells.
Correlation/correlatedxe2x80x94refers to the correlation between the measured signal and the parameter which is to be determined. Such correlation may be manifested either by a proportional increase in the signal in line with the level of said parameter, or a proportional decrease in the signal in line with said parameter.
Growth statusxe2x80x94a term referring particularly to a culture of cells. A growth status may be an algorithmic growth phase of the culture, a stationary phase, etc.
Determination/determiningxe2x80x94including a qualitative determination of existence or of non-existence of a certain parameter or condition as well as a qualitative determination of the level of such parameter or condition. The parameter which is determined may for example be the existence of the certain substance in the medium or cell as well as a quantitative determination of its level.